Registration Dossier

Administrative data

Description of key information

Oral: NOAEL (28 days, rat) = 250 mg/kg bw/day (OECD 422, GLP, target organ: kidney, male rat specific)
Inhalation: NOAEC (1 year, rat) = 1600 mg/m³ (target organ: kidney, male rat specific)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1-2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1 600 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral

A GLP-compliant Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD 422) was conducted with methylcyclohexane (MHLW, 2011). Groups of 12 male and 10 female rats per dose were given methylcyclohexane by oral gavage at 62.5, 250 and 1000 mg/kg bw/day, daily for 28 days. Concurrent male and female vehicle (corn oil) control groups were included. Satellite control and treatment groups were included for a 14-day recovery period. These groups comprised 6/12 males in the control and all dose groups, and 5/10 females in the control and high-dose groups, respectively.

No mortality occurred during the study period. Transient salivation following dosing was observed in all males and females of the 1000 mg/kg bw/day groups. No or no toxicologically relevant effects were seen on body weight, food and water consumption, detailed clinical observations, FOB, urinalysis and levels of thyroid-related hormones.

No changes in haematological parameters were found in males after the administration period. After the recovery period, MCHC and WBC values were decreased in males. The toxicological relevance of this finding is questionable since such a tendency was not observed at the end of the administration period and the corresponding control value was clearly lower than the one after the recovery period. A significant increase in reticulocytes and monocytes was observed in females of the 1000 mg/kg bw/day group after the administration period, but not after the recovery period.

Total cholesterol was significantly increased in males of all treated groups after the administration period without clear dose-response relationship. ALT, gamma-GT, total protein and Ca values were significantly increased, and Cl significantly decreased, in males at 1000 mg/kg bw/day after the administration period. Cl values were slightly but significantly decreased at 250 mg/kg bw/day. After the recovery period, only ALT and total cholesterol were still significantly increased in males dosed 1000 mg/kg bw/day. Glucose was significantly decreased in females of the 1000 mg/kg bw/day group after the administration period. No changes were found in females of the satellite recovery groups.

In males at the dose level of 1000 mg/kg bw/day, absolute and relative weight of liver and kidney were significantly increased after the administration period. Relative liver weight was slightly (by 8.5%) but significantly increased in males dosed 250 mg/kg bw/day after the administration period. No differences in liver and kidney weights were observed after the recovery period at any dose level. In males of the 1000 mg/kg bw/day group, relative testis weight was significantly increased after the recovery period. However, this change was not considered as test item-related, since this was not observed after the administration period.

Based on the results of clinical chemistry examinations and organ weights, the test item may affect the liver. However, no changes were found at histopathological examinations in the liver. The test item did not cause any organic change. Changes observed in kidney weight and at histopathological examination suggest that the test item or its metabolites impaired lesions which occurred naturally. However, these changes in kidneys were reversible since they disappeared after the recovery period.

In females dosed 1000 mg/kg bw/day, absolute and relative weights of adrenals as well as relative weight of liver, kidney and uterus were significantly increased after the administration period. The increase in relative uterus weight was not considered to be test item-related, since this change was within the historical background data of the test facility. After the recovery period, the absolute (but not the relative) liver weight was significantly increased and no further changes were found.

One male in the control group showed bilateral small size of testis and bilateral softness of testis after the administration period. One male in the 62.5 mg/kg bw/day showed lateral small size of testis and lateral small size of epididymis after the administration period. One male in the 1000 mg/kg bw/day showed bilateral small size of testis and bilateral small size of epididymis after the administration period. Due to the low incidence and lack of dose-response relationship, these changes were considered accidental. No abnormalities were found in females after the administration period and in both sexes after the recovery period at gross necropsy.

After the administration period, one male in the control group showed slight cellular infiltration in the heart. One male in the control groups showed slight extramedullary haematopoiesis in the spleen. Slight bilateral hyaline droplet in renal tubule was observed in all males at 1000 mg/kg bw/day and 4 males at 250 mg/kg bw/day. Changes at 1000 mg/kg bw/day were statistically significant and dose-dependent. Slight bilateral basophilic change in renal tubules was observed in one male at 250 mg/kg bw/day. One male in the control group showed lateral cyst in kidney. Atrophy of the seminiferous tubule was observed in one male each at 62.5 and 1000 mg/kg bw/day. These animals showed also small size or softness of testis at gross pathology. One animal in the control, 62.5 and 1000 mg/kg bw/day group, respectively, showed slight cell debris in the lumen of epididymis. These animals showed also small size or softness of epididymis at gross pathology. Cell infiltration in the ventral prostate was observed in one male of the 1000 mg/kg bw/day group. All these changes except for slight bilateral hyaline droplet in the renal tubule at 250 and 1000 mg/kg bw/day were considered accidental, since they are usually observed in untreated animals and the incidence was not statistically significant. Hyaline droplet formation was likely related to alpha2µ-globulin accumulation, which is an effect specific for male rats and not relevant for humans.

After the recovery period, two males in the 1000 mg/kg bw/day group and one male in the control group showed bilateral/lateral basophilic change in the renal tubules. One male in the control group showed lateral cyst in the kidney. These changes were considered accidental, since they are usually observed in untreated animals and the incidence was not statistically significant.

One female of the control and 1000 mg/kg bw/day groups, respectively, showed slight lateral retinal dysplasia in the eyeball. This change was not considered test item-related, since it was also observed in control group and the incidence was not statistically significant.

Based on the effects observed at 1000 mg/kg bw/day, in particular transient salivation after dosing (males and females) and non-reversible increases in ALT and total cholesterol values in males and relative liver weight in females, the oral NOAEL for males and female rats was 250 mg/kg bw/day.

The subacute oral toxicity of methylcyclohexane was investigated in a study conducted according to OECD guideline 407 and in compliance with GLP (NITE, 2001). Methylcyclohexane (diluted in corn oil) was administered by gavage to groups of rats (5 per sex and dose) at 100, 300 and 1000 mg/kg bw/day, 7 days/week for 28 days. A control group (5 males and 5 females) was concurrently treated with the vehicle. Satellite control (vehicle) and high-dose groups consisting of 5 animals per sex and dose were included in the study for a recovery period of 14 days post-treatment.

No mortality was observed. Transient salivation was observed in males at 300 mg/kg bw/day and in both sexes at 1000 mg/kg bw/day within one hour after administration. This change was not observed during recovery period.

No treatment-related changes were observed in either sex in the functional observation battery, haematological and urinalysis examinations.

An increase in the total protein, a decrease in the alkaline phosphatase and a decrease in the albumin/globulin ratio (A/G) were found in males at 1000 mg/kg bw/day after both treatment and recovery period. These changes were considered treatment-related but the mechanism was not clear. An increase in total cholesterol and a decrease in alanine aminotransferase were observed in females in the 1000 mg/kg bw/day group. These effects were reversible and regarded as a compound-related effect but the mechanism was not clear.

An increase in absolute and relative weight of the liver was observed in males and females of the 1000 mg/kg bw/day group. This change was one of the toxicological effects of test compound based on the results of histopathology.

No treatment-related changes were observed in either sex at gross pathology examination. Changes noted in individual males (at 100 and 300 mg/kg bw/day) and females (all treatment and high-dose recovery groups) were sporadic, and thus showed no dose-related increase in incidence or severity. Findings included: black or brown patch in the lung; nodule in the large intestine; brown patch, nodule and white patch in the liver; cyst in the kidney; dilated lumen in the uterus. 

At histopathological examination, hypertrophy in hepatocytes was observed in 5 males and one female of the 1000 mg/kg bw/day group and this result correlates with the result of organ weights. This change was reversible since it was not seen after recovery period. This change was not significant in females. It seems that there is sex specificity. Hyaline droplet degeneration of the kidney was observed in one male at 100 mg/kg bw/day and in all 5 males at 300 and 1000 mg/kg bw/day, respectively. There was a statistically significant difference in the mid- and high-dose groups. This effect was reversible, since slight hyaline droplet degeneration was seen in only one male of the high-dose recovery group. Hyaline droplet formation was also observed in two females in the 1000 mg/kg bw/day group and all females of the 1000 mg/kg bw/day recovery group. This change was regarded as a compound-related effect. It was supposed that the test compound was bound with alpha2µ-globulin and this change was reversible in males. On the other hand, this change was not reversible in females but occurred more slowly than in males. This finding was unusual, since alpha2µ-globulin-mediated kidney effects of hydrocarbons are generally considered to be specific for male rats and without relevance for humans.

In conclusion, based mainly on changes found in clinical signs, clinical chemistry, organ weight and histopathological examinations (transient salivation, increased total cholesterol, decreased alanine aminotransferase, increased absolute and relative liver weight, enlarged hepatocytes and hyaline droplet formation) the oral LOAEL for female rats was considered to be 1000 mg/kg bw/day. For males, due to transient salivation, increased body weight and food consumption and the occurrence of hyaline droplet degeneration in 5/5 animals, the oral LOAEL was 300 mg/kg bw/day.

No adverse effects were observed in females given 300 mg/kg bw/day. Thus, this value was considered the oral NOAEL in females. Apart from a slight hyaline droplet degeneration seen in 1/5 animals, no other toxicologically relevant effects were seen in males at 100 mg/kg bw/day. Kidney effects were reversible in males, as only slight hyaline droplet degeneration in this organ was seen in one animal at histopathological examination of the 1000 mg/kg bw/day recovery group. Therefore, the oral NOAEL for males was 100 mg/kg bw/day.

In accordance with Regulation (EC) No 1272/2008, Annex 1: 3.9.2.9.5 and 3.9.2.9.7, the oral LOAEL (male) of 300 mg/kg bw/day determined in this study falls within the guidance value range for classification for Specific target organ toxicity – repeated exposure (STOT-RE) Category 2, after applying a factor 3 for time extrapolation for a 28-day study. However, according to Regulation (EC) No 1272/2008, Annex 1: 3.9.2.8.1, in particular items (a) and (e), the observed clinical signs and changes in body weight and food consumption as well as the alpha-2-microglobulin-mediated, male rat specific hyaline droplet degeneration in the kidney do not justify classification, as described in ECHA’s Guidance on the Application of the CLP Criteria (Version 2.0, April 2012). Thus, methylcyclohexane does not fulfil the classification criteria for toxicity after repeated exposure according to Regulation (EC) No 1272/2008 and Directive 67/548/EEC.

In another study conducted with focus on urinary metabolites, a group of 8 male rats was given undiluted methylcyclohexane by oral gavage at 800 mg/kg bw, every other day for 14 days (Parnell et al., 1988). A control group of 6 animals was treated with 800 mg/kg bw of water. Examinations included only daily determination of body weight, collection of urine samples for metabolite analyses during the first 48 h and histopathological examination of kidney sections.

The selected dose level was expected to induce nephropathic lesions, and therefore, kidney tissues from treated rats were compared to controls for characteristic lesions of hydrocarbon-induced nephropathy, including hyaline droplet formation, tubular cysts and papillary calcification. However, histopathological examination of the rat kidney slices indicated only very slight traces of nephropathy.

Inhalation

Inhalation is the most relevant route of exposure for methylcyclohexane, and several studies have been conducted to investigate the potential toxic effects following subchronic and chronic exposure to methylcyclohexane vapours in different species.

The most comprehensive studies were reported by Kinkead et al. (1985), in which rats, mice, hamsters and dogs were used for investigations.

Groups of rats (65 per sex and group), mice (200 females per group), hamsters (100 males per group) and dogs (4 per sex and group) were whole body-exposed to air or methylcyclohexane concentrations of 400 and 2000 ppm (corresponding to ca. 1600 and 8000 mg/m³), 6 h/day, 5 days/week, for 12 months. At the end of the exposure period, 10 rats per sex, 20 mice and 10 hamsters per group, respectively, were sacrificed and subjected to necropsy. The remaining animals were maintained for a post-exposure observation period of further 12 months or 5 years (only dogs).

For the rat study, no clinical signs were reported and mortalities (number, time and cause of death) were not explicitly mentioned. From the number of animals used for histopathological examinations, it can be deduced that 1 male and 1 female in the control and 1 male in the 8000 mg/m³ group died during the 12-month exposure period.

Male rats exposed to both levels of the test material showed depressed growth throughout the study period. At the end of the 12-month exposure period, the mean body weight of the test animals was decreased by about 7% compared with the control group. Although the male rats showed an increase in weight gain after removal from the exposure chambers, they still did not attain the mean weight of the unexposed control group. At this time point, the mean body weight of the treated animals was decreased by ca. 4% when compared with the control animals. Despite the differences in mean body weight values between control and treated groups at each time point, the body weight gain rate was comparable among groups from Month 2-12 of exposure. Due to the lack of concentration dependence and of information on statistical significance, this effect was considered non-adverse. The female rat weights were unaffected during exposure as well as during the post-exposure observation period.

Haematological and clinical chemistry analyses showed no biologically significant differences between rats exposed to the test material and control rats. Statistically significant differences from control values were seen in white blood cell count (WBC) in both male groups (both ca. 20% decrease) and in the 8000 mg/m³ female group (ca. 30% decrease). Males in the 1600 mg/m³ group showed slight but statistically significant changes in haematocrit value (2% increase), potassium level (13% increase), sodium level (2% decrease) and creatinine (16% increase). In 8000 mg/m³ males, sodium levels were also statistically significantly decreased by ca. 2%. Because of haemolysis in most samples of female rat blood, no clinical chemistry comparisons could be made.

Histopathological examination of tissues from animals that were sacrificed at the end of the 12-month exposure as well as from those which died during the exposure period showed no differences between female control and exposure groups. In males rats, a statistically significant increase in the incidence of testicular tumours was observed in the 1600 mg/m³ group (control: 0/11; 1600 mg/m³: 5/10; 8000 mg/m³: 2/11). The authors stated that tumours seen in the male rats are commonly found in this strain. A dose-related but not statistically significant increase in the incidence of renal tubular dilatation was observed (controls: 1/11; 1600 mg/m³: 2/10; 8000 mg/m³: 4/11). No other indication of kidney injury was seen at the end of the 12-month exposure.

At the end of the 12-month post-exposure period, no statistically significant differences in the incidences of neoplastic and non-neoplastic lesions were seen between female control and exposure groups. Only one tumour, a benign endometrial stromal polyp, was found in any female rat and this was seen in an animal exposed to 1600 mg/m³. In male rats, the major target organ was the kidney where two types of lesions were associated with exposure. Virtually all of the male rats had lesions consistent with progressive renal nephropathy, common in older male rats. In the male rats exposed to 8000 mg/m³, there was a statistically significant increase in the occurrence of medullary mineralization (control: 1/53; 1600 mg/m³: 2/55; 8000 mg/m³: 19/52) and epithelial hyperplasia of the renal papilla (control: 1/53; 1600 mg/m³: 1/55; 8000 mg/m³: 23/52). Interstitial cell tumours of the testes, seen at study termination, appeared to be equally distributed between the test and control groups and not related to exposure. In general, neoplastic changes seen in rats were those expected in aging animals of this species. According to the authors, statistical analysis of the data failed to indicate any significant increase in tumour formation in the exposed animals when compared to the controls.

In conclusion, based on the progressive renal nephropathy observed at histopathological examination of male rats, 8000 mg/m³ (2000 ppm) was considered the LOAEC. Thus, in this study, 1600 mg/m³ (400 ppm) was identified as the NOAEC, at which only a non-adverse depression of body weight was observed in male rats.

For mice, no clinical signs and body weight data were reported and mortalities (number, time and cause of death) were not explicitly mentioned. From the number of animals used for histopathological examinations, it can be deduced that 9/200 animals in the control group, 15/200 in the 1600 mg/m³ group and 19/200 animals in the 8000 mg/m³ group died during the 12-month exposure period.

Histopathological examination revealed no statistically significant changes in animals sacrificed at the end of the 12-month exposure period as well as in animals which died during this time. The results of examination of tissue from the animals that died during the post-exposure observation period or were killed at the study termination showed no significant lesions when compared to the control group. However, increased incidences in uterine multiple cysts were reported (controls: 10/164; 400 ppm: 22/158; 2000 ppm: 23/152). Lesions noted were stated to be those commonly seen in older animals of this species. Neoplastic changes seen in mice after the 12-month post-exposure period were stated to be those expected in aging animals of this species. According to the authors, statistical analysis of the data failed to indicate any significant increase in tumour formation in the exposed animals when compared to the controls.

Based on the lack of significant adverse effects, a NOAEC (mouse, female) of 8000 mg/m³ was identified in this study.

For hamsters, no clinical signs were reported and mortalities (number, time and cause of death) were not explicitly mentioned. From the number of animals used for histopathological examinations, it can be deduced that 14/100 animals in the control group, 10/100 in the 1600 mg/m³ group and 7/100 animals in the 8000 mg/m³ group died during the 12-month exposure period.

A definite depression in mean body weights was seen in the exposed hamster groups but it was not clearly related to the exposure concentration throughout the 12-month exposure period. At the end of the exposure period, the group mean body weight was decreased by about 11% at 1600 mg/m³ and by about 15% at 8000 mg/m³.

For both exposure and control groups, body weights increased until about Month 2 but decreased gradually from Month 4 onwards. Immediately following exposure, both exposed hamster groups gained weight and became equivalent to the control group at Month 14. Thereafter, body weights decreased again, those of the animals having been exposed to 1600 mg/m³ more than those of the 8000 mg/m³ group.

At the end of the 12-month exposure period, histopathological examinations showed lesions that were fairly uniform in all groups and not related to exposure. Only one tumour, a benign tumour of an adrenal gland in an animal from the high exposure group was seen in all hamsters examined. Following the 12-month post-exposure period, there were no statistically significant differences in the incidences of neoplastic and non-neoplastic lesions between control and exposure groups, although increased incidences in renal cortical fibrosis were observed (controls: 4/75; 1600 mg/m³: 12/76; 8000 mg/m³: 10/81). Lesions noted were stated to be those commonly seen in older animals of this species.

In this study, a NOAEC could not be identified. Due to the significantly depressed body weight during the exposure period and at the end of the post-exposure period, the LOAEC (hamster, male) was 1600 mg/m³.

For dogs, no data were reported on mortality, clinical signs or body weight. Clinical determinations on dog blood taken at biweekly intervals gave variable but not treatment-related results. The only parameter affected by exposure to methylcyclohexane was a transient increase in the mean SGPT level of the dogs exposed to 8000 mg/m³ which was caused by a single dog exhibiting a high SGPT level during the seventh exposure week while the other animals of the group were normal. The increase in SGPT values occurred again between 39 and 43 weeks in the same dog from the 8000 mg/m³ group as well as in two dogs from the 1600 mg/m³ group. According to the authors, significant differences in SGPT values of dogs were not seen during the five-year post-exposure period.

No neoplastic and no significant non-neoplastic lesions were found in dogs when compared to the control group. Lesions noted were stated to be those commonly seen in older animals of this species.

Due to the lack of significant adverse effects, a NOAEC (dog) of 8000 mg/m³ was identified in this study.

In an earlier study, rabbits (4/group) were exposed 6 h/day, 5 days/week to methylcyclohexane concentrations of 948 (for 10 weeks), 4570 (for 10 weeks), 11350 (for 3 weeks), 21900 (for 4 weeks), 28750 (for 2 weeks), 39550 (for 2 weeks) and 59900 (for up to 70 min) mg/m³ (Treon et al., 1943b).

At the highest concentration, all animals died within 70 min after exposure initiation. Prior to death, animals showed severe convulsions, rapid narcosis, laboured breathing, salivation and conjunctival congestion. After 2 weeks of exposure to 28750 and 39550 mg/m³, 1/4 and 4/4 animals died, respectively. At 39550 mg/m³, convulsions, light narcosis, laboured breathing, salivation and conjunctival congestion were observed, while animals exposed to 28750 mg/m³ showed lethargy and impaired coordination of legs. Only slight lethargy was noted in animals exposed to 21900 mg/m³ and no clinical signs were seen at 948 to 11350 mg/m³.

Animals exposed to non-lethal concentrations (0.948-21.90 mg/L) showed no adverse effects in body weight gain, while animals exposed to lethal concentrations lost weight: 39 and 390 g after a 2 week exposure to 28.75 and 39.55 mg/L, respectively. No effects were seen in haemoglobin concentration, erythrocytes and leucocytes counts. Results from histopathological examinations were reported only for animals exposed to 11350 mg/m³. Barely demonstrable evidence of cellular liver and kidney injury (not further specified) was observed.

Based on histopathological findings, the (3-week) subacute LOAEC was 11350 mg/m³, in this study. The (10-week) subchronic NOAEC was considered to be 4570 mg/m³ due to the lack of effects on the parameters examined.

Within the same study by Treon et al. (1943b), one Rhesus monkey was exposed to methylcyclohexane at 1460 mg/m³, 6 h/day, 5 days/week for 10 weeks. At the end of the exposure period, body weight was increased by 454 g. No clinical signs, no effects in haemoglobin concentration, erythrocytes and leucocytes counts and no microscopic lesions were noted.

In another report, rats exposed to technical grade methylcyclohexane (70% pure) at 11000 mg/m³, 6 h/day for 5 consecutive days showed symptoms of anaesthesia during exposure. The animals recovered after each exposure and no mortality occurred (Sutton, 1969). No further details on methods and results were provided.

Dermal

Methylcyclohexane was applied to the clipped skin of one rabbit in twelve 5 ml portions at 5 min intervals within 1 h, after which the material was removed by washing with soap and water (Treon et al., 1943a). This uncovered skin procedure was done on 6 consecutive days. Based on the reported total amount of test substance applied daily (60 mL), the specific gravity (0.767) and the animal body weight (3.185 kg), the applied dose was roughly equivalent to 14450 mg/kg bw/day. The test substance was applied on a surface of approximately 24 square inches (corresponding to ca. 155 cm²). Thus, the local dose was about 300 mg/cm²/day.

Slight hypothermia and a slight loss in body weight (no further details given) were observed in the treated animal during the 6-day study period. The loss in body weight was regained within 2 days. Repeated application of methylcyclohexane to the skin of a rabbit induced local irritation and thickening. The irritation appeared on the second day and increased with successive treatments. Hardening of the skin, thickening and ulceration appeared later and the experiment was terminated after the 6th day.

Other routes: subcutaneous

Methylcyclohexane was tested in a subchronic toxicity study focusing on potential compound-related effects on the reproductive system (Kim et al., 2011). Groups of 5 male and female rats were exposed to methylcyclohexane at 10, 100 and 1000 mg/kg bw/day by subcutaneous injections, 5 days/week for 13 weeks. Concurrent vehicle control animals were treated with olive oil.

Mortality was observed in 4/5 female and 4/5 male rats receiving 1000 mg/kg bw/day (between Day 5 and 60 of the study period). No mortality was observed in the other treatment groups and in the controls. However, a slower movement was noted in animals of the low- and mid-dose. No further treatment-related abnormalities were found in the surviving animals.

Body weight was clearly and statistically significantly decreased in both male and female animals at 1000 mg/kg/day.

No effects on haematological parameters were seen in male animals dosed 10 and 100 mg/kg bw/day. In females of the 100 mg/kg bw/day group, monocytes were statistically significantly decreased. However, this change was not considered to be of biological relevance, since it was within the normal range of this parameter in rats. The number of surviving animals in the 1000 mg/kg bw/day male and female groups was too small for assessment of statistical significance in the observed changes. These included a marked increase in the counts of white blood cells, neutrophils, eosinophils and platelets observed in the surviving male, whereas the red blood cell count, haemoglobin, and haematocrit were decreased. In contrast to the finding in the surviving male of the 1000 mg/kg bw/day group, all haematological parameters were decreased in the surviving female rat of this group. The changes in animals at 1000 mg/kg bw/day were considered to be treatment-related.

Inorganic phosphorus (IP) concentrations were slightly but statistically significantly and dose-dependently increased in the 10 and 100 mg/kg bw/day male groups. At 1000 mg/kg bw/day, IP concentration was also higher than the mean concentration in the control group. In females, no significant changes in IP concentration were observed, but a dose-dependent tendency to decrease. Males in the 100 mg/kg bw/day showed a very slight but statistically significant increase in total protein and albumin in serum. These values decreased again in the surviving male dosed 1000 mg/kg bw/day. These changes were considered not to be treatment-related as they were slight and there was no dose dependency. Urea nitrogen in blood (BUN), creatinine and alkaline phosphatase were markedly increased in females of the 100 mg/kg bw/day group. However, due to high interindividual variability, these changes were not statistically significant. At 1000 mg/kg bw/day, the low number of surviving animals did not allow statistical analysis. Male animals showed an increase in ALP, creatine phosphokinase, and IP. Female animals showed an increase in lactate dehydrogenase and ALP and a decrease in BUN, alanine aminotransferase, and IP.

No changes in organ weights were observed in male and female animals dosed at 10 and 100 mg/kg bw/day. The weight of thymus, adrenal gland, lungs, kidney, spleen, liver and brain was decreased in males of the 1000 mg/kg bw/day group. In females of the same group, a decrease was observed in the weight of thymus, adrenal gland, ovary, lungs, kidney, spleen and liver.

Microgranuloma and bile duct proliferation were observed in each 1/5 males at 10 mg/kg bw/day. Hepatic cytoplasmic vacuolation was observed in 1/5 males at 100 mg/kg bw/day. At 1000 mg/kg bw/day, liver microgranuloma, bile duct proliferation, cardiac hyperemia/hemorrhage and myocardial necrosis were observed (incidence not reported). Some of the animals showed renal protein casts and hyperemia/haemorrhage (incidence not reported). No effects were observed in the diameter of the seminiferous tubules and thickness of gametes. In females of the 100 mg/kg bw/day, liver microgranuloma and bile duct proliferation were observed in 1/5 and 2/5 animals, respectively. At 1000 mg/kg bw/day, bile duct proliferation of the liver, hyperemia, and renal hyperemia/hemorrhage were seen, but the incidence was not reported. Due to the low incidence of liver changes in the 10 and 100 mg/kg bw/day groups and the fact that such changes are commonly found also in untreated rats, these findings were considered to be spontaneous and not treatment-related. According to the authors, due to the severe level of abnormalities seen at 1000 mg/kg bw/day, findings in organs of male and female animals seen at this dose level were considered to be treatment-related.

No effects were observed in spermatogenic cell count and morphology at any dose level. In males, no effects were seen on the levels of prolactin, testosterone, follicle stimulating hormone and luteinizing hormone. A very slight but statistically significant and dose-dependent increase in estradiol levels was observed at 10 and 100 mg/kg bw/day. At the same dose levels, a significant decrease in progesterone levels was noted. The toxicological relevance of this finding is unclear, since no corresponding alterations were observed in testes at microscopic examination. No female data were reported. According to the authors, the hormone levels in females were dramatically changed in accordance with the estrous cycle. The menstrual cycle period was statistically significantly increased in the 4th week in the 10 mg/kg bw/day group, but this change was not dose-dependent. After the 13th week, no effects were observed.

Based on mortality, decreased body weight, changes in haematological and clinical chemistry parameters and histopathological findings, the LOAEL was set at 1000 mg/kg bw/day and the NOAEL for males and females was 100 mg/kg bw/day.

In summary, sufficient, adequate and reliable data are available for assessment of the potential toxicity of methylcyclohexane via the oral and inhalation routes; the latter being the most relevant route of exposure for methylcyclohexane.

For the purpose of chemical safety assessment, the two most reliable studies investigating the subacute oral toxicity were considered together. In both studies, groups of male and female rats were orally exposed to methylcyclohexane for a period of 28 days, followed by a 14-day recovery period. The first study resulted in a NOAEL of 250 mg/kg bw/day and a LOAEL of 1000 mg/kg bw/day for both male and female rats (MHLW, 2011). The second study identified NOAEL values of 100 (males) and 300 (females) mg/kg bw/day and LOAEL values of 300 (males) and 1000 (females) mg/kg bw/day, respectively (NITE, 2001). While the MHLW (2011) study used more male animals per dose and included recovery groups of the control, low- mid- and high-dose male and high-dose female groups, the NITE (2001) study comprised only control and high-dose recovery groups.

The LOAEL of 300 mg/kg bw/day from the NITE (2001) study was mainly based on transient salivation and hyaline droplet degeneration in the kidney after the treatment period; the latter being reversible at 1000 mg/kg bw/day after the recovery period. In the MHLW (2011) study, transient salivation was not observed at 250 mg/kg bw/day and hyaline droplet formation after the treatment period was shown to be reversible both at 250 and 1000 mg/kg bw/day.

Taken together, the subacute oral NOAEL of methylcyclohexane in male rats is below 300 and above 100 mg/kg bw/day. The MHLW (2011) study provides robust evidence that the subacute oral NOAEL in male rats is most likely to be 250 mg/kg bw/day. Therefore, this value was selected as dose descriptor and point of departure for hazard and risk assessment of methylcyclohexane via the oral and (after route-to-route extrapolation) the dermal routes.

The most comprehensive and reliable study investigating the potential toxicity of repeated exposure to methylcyclohexane by inhalation was reported by Kinkead et al.(1985). After a (12-month) chronic exposure to methylcyclohexane (6 h/day, 5 days/week) followed by a 12-month post-exposure observation period, a NOAEC of 1600 mg/m³ was identified for male rats. The fact, that exposure related renal injury was strictly limited to male rats and that the test substance belongs to a category of substances which are known for their ability to induce alpha2µ-globulin-related nephropathy in male rats, the observed effects in the kidneys have to be regarded as species-specific and are not relevant for risk assessment in humans. Therefore, the selection of this dose descriptor for the purpose of chemical safety assessment is considered a worst-case and conservative approach.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The selected study is the most adequate and reliable study based on overall assessment of quality, duration and basis of the dose descriptor level. The dose descriptor provided in this study is the highest available NOAEL below the lowest available LOAEL (refer to the endpoint discussion for further details).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The selected study is the most adequate and reliable study with the longest duration and the lowest dose descriptor.

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: kidneys

Repeated dose toxicity: inhalation - systemic effects (target organ) urogenital: kidneys

Justification for classification or non-classification

The available data on the repeated dose toxicity of methylcyclohexane do not meet the criteria for classification according to Regulation (EC) No 1272/2008 or Directive 67/548/EC, and are therefore conclusive but not sufficient for classification.